CN107835817A

CN107835817A - The ester of steroidal lactams and double (2 chloroethyl) amino-benzene oxygen propanoic derivatives

Info

Publication number: CN107835817A
Application number: CN201680038078.XA
Authority: CN
Inventors: D·特拉法里斯
Original assignee: Energy Biotechnology Co; Green Nica Joint-Stock
Current assignee: Energy Biotechnology Co; Green Nica Joint-Stock; Galenica SA
Priority date: 2015-06-29
Filing date: 2016-06-28
Publication date: 2018-03-23
Anticipated expiration: 2036-06-28
Also published as: AU2016286205B2; RU2018103071A3; BR112017027850B1; TR201902369T4; IL256456A; IL256456B; KR20180015266A; CA2990307A1; CN107835817B; ES2712429T3; AU2016286205A1; US20180194800A1; CA2990307C; KR102647748B1; PT3186268T; AU2016286205B8; WO2017001439A1; MX369930B; HRP20190327T1; SI3186268T1

Abstract

With new high aza sterides esters, its preparation method, the pharmaceutical composition comprising them and its purposes in treating cancer of the derivative for being alkylated double (2 chloroethyl) amino-benzene oxygen propionic acid and substitution.

Description

The ester of steroidal lactams and double (2- chloroethyls) amino-benzene oxygen propanoic derivatives

Invention field

The present invention relates to new height-azepine-steroidal esters with being alkylated mustargen, described alkylation mustargen is aniline Derivative, such as double (2- chloroethyls) amino-benzene oxygen propionic acid and the derivative of substitution.

Background of invention

At present, in Present clinical practice, the alkylation anticancer as mustargen is still effective a kind of antineoplastic Thing, its therapeutic effect from its make alkyl be connected to cell DNA and produce notable DNA damage ability (Hurley LH, Nature Rev Cancer,2002,2：188-200；Brendel M and Ruhland A, Mutat Res, 1984；133：51- 85)。

Steroidal conjugate was used as to the carrier of cytotoxicity alkylating agent in the past, because they reduce system toxicity and changed The effect of being apt to cancer therapy (Wall ME et al., J Med Chem, 1969,12：810-8；Catane R,Cancer Treat Rep,1978；62：1264-5).As Estramustine (ester of estradiol and mustargen) and prednimustine (prednisolone and benzene fourth The ester of sour mustargen) steroidal alkylating agent be applied to cancer in terms of row gland cancer and lymphoproliferative malignant tumour before the treatment respectively at present In disease therapy (Catane R, Cancer Treat Rep, 1978,62：1264-5；Matsumoto K et al., Med Oncol, 2013,30：717；IARC Monogr Eval Carcinog Risks Hum,1990,50：115-22；Hiddemann W, Eur J Cancer,1995,31A(13-14)：2141-5).

In view of acute and general toxicity caused by these medicines reduces, with their alkylation composition individually caused by it is high More toxicity are not on the contrary, their active anticancer obtains too big improvement, and although initial evaluation but their target cancer cells Specificity quite lack.However, even if Estramustine and prednimustine play the main molecules pharmacology machine of active anticancer System differs markedly from the specific action to steroid receptors, it is however generally that they show in clinical practice, and good and what is improved controls Treat effect.

A few class height-azepine-steroidal esters or lactams steroidal ester (the steroidal ring being conjugated with alkylating agent had been synthesized in the past In contain lactams group-NHC=O- steroidal compounds), and test it in vitro and in vivo in preclinical environment Toxicity and active anticancer (Wampler GL and Catsoulacos P, Cancer Treat Rep, 1977,61：37-41； Catsoulacos P and Catsoulacos D, Anticancer Res, 1991,11：1773-7；Catsoulacos P and Catsoulacos D,Anticancer Res,1993,13(4)：1203-8；Catsoulacos P et al., Oncology, 1994,51：74-8；Catsoulacos P and Catsoulacos D, Anticancer Res, 1994,14 (6B)：2525-8； Camoutsis C and Trafalis DT, Invest New Drugs, 2003,21：47-54；Koutsourea Al et al., Bioorg Med Chem,2008,16：5207-15).

Lactams steroidal alkylation esters show that they produce significantly reduced acute toxicity in vivo, and they are in vitro Confirm in vivo enhancing and desired antitumor activity as rich as Croesus, and each unmodified (non-lactams) steroidal alkane Agent produces significantly lower active anticancer to corresponding experimental tumor system or does not almost have active anticancer.Except producing cell Beyond DNA damage, the molecular pharmacology mechanism for significantly improving the antitumaous effect of lactams steroidal alkylation esters is still indefinite.This Outside, the biologic importance for the position that one or more lactam groups are incorporated into steroidal structure is also unknown.It is in addition, logical Cross the alkylating agent that ester bond is conjugated on lactams steroidal and play remarkable effect, and adjust the ratio of acute toxicity and antitumor activity Example, thus adjust the degree for the treatment of ratio caused by lactams steroidal alkylating agent.Up to the present, synthesized and tested several Kind effective lactams steroidal alkylating agent, but those with compared with high anti-tumor activity are shown with larger toxicity, and demonstrate,prove Those activity of real low toxicity are relatively low.These observations show, the effective lactams steroidal alkylation for developing and producing new is sewed There is obvious demand in compound, lactams steroidal alkylation conjugate produces optimal low toxicity and compared with high anti-cancer activity, because This produces optimal therapeutic index.

The research on the lactams steroidal alkylation esters of nitrogen mustard derivatives showed in the past, the alpha-amido of 3 beta-hydroxy-13- - 5 α of 13,17- open loops-androstane -17- acid -13,17- lactams-[p- [two (2- chloroethyls) amino] phenyl] acetic acid esters (ASE, NSC-290205) is generated very in preclinical test to internal acute toxicity and internal and external antitumor activity Good counterbalance effect, maintains significantly high therapeutic index.

Therefore, ASE represents the recruit for developing same type medicament and tests their therapeutic efficiencies compared with ASE " gold " standard.

Summary of the invention

The present invention provides the new esters of steroidal lactams and alkylating agent.More specifically, the compound of the present invention is steroidal The esters of lactams and double (2- chloroethyls) amino-benzene oxygen propanoic derivatives.The lactams steroid of these compounds and prior art Body alkylation esters, which are compared, shows higher antitumor activity and lower acute toxicity, and can be used as antineoplastic and cancer Remedies.

Detailed description of the invention

The compound or its pharmaceutically acceptable salt of offer formula (I) of the present invention：

Wherein

R₁Selected from the group being made up of following group

R₂Selected from the group being made up of following group：H、-CH₃,-CH=CH₂、-CH₂-CH₃、-CH₂CH₂CH₃、

R₃Selected from by H ,-OH ,-NH₂The group of composition.

Preferably, R₁Selected from the group being made up of following group

It is highly preferred that R₁Selected from the group being made up of following group

Preferably, R₂Selected from by H ,-CH₃,-CH=CH₂、-CH₂-CH₃、-CH₂CH₂CH₃The group of composition.It is highly preferred that R₂It is H。

Preferably, R₃It is H or NH₂。

The hydroxyl of double (2- chloroethyls) amino-phenol parts of the compound of formula (I) can be in the amino relative to phenyl ring On ortho position, meta or para position.

The compound of formula (I) includes at least one asymmetric center.Do not clearly stating the three-dimensional of asymmetric center In the case of, the structure is intended to all individually stereoisomer and their mixtures.

The compound of formula (I) includes at least one basic functionality, therefore can form medicine by using suitable acid treatment Acceptable salt on.Suitable acid includes pharmaceutically acceptable inorganic acid and pharmaceutically acceptable organic acid.Pharmaceutically The example of acceptable salt includes hydrochloride, hydrobromate, sulfate, phosphate, nitrate, acetate, propionate, butyric acid Salt, maleate, fumarate, tartrate, citrate, lactate, oxalates, succinate and benzoate.

The compound of formula (I) or their pharmaceutically acceptable salt can be used for treating extensive cancer.Preferably, it Be used for treat oophoroma, breast cancer, prostate cancer or leukaemia.

Compared with the lactams steroidal alkylation esters of prior art, the compound of formula (I) or they can pharmaceutically connect The salt received shows higher antitumor activity and lower acute toxicity, can be used as antineoplastic and cancer therapeutic agent.Hereafter The preclinical test for biological activity disclosed in embodiment is incorporated to manner of comparison, and purpose be to show it is described new Superiority of the alkylation lactams steroidal esters in terms for the treatment of of cancer effect, two positive controls, i.e. alkylating agent (3- (4- (double (2- chloroethyls) amino) phenoxy group) propionic acid, pBCEAPOPA) the independent experimental lactams steroidal alkanisation with the classification " gold " the standard ASE (NSC-290205) of agent.

The present invention also provides the compound comprising formula (I) or the pharmaceutical composition of its pharmaceutically acceptable salt.This kind of medicine Compositions can be formulated for applying by any appropriate approach, for example, orally, intranasal, part or parental routes. For example, pharmaceutical composition can be prepared piece agent, capsule, pulvis, solution, supensoid agent, cream or gel.Except formula (I) compound or its pharmaceutically acceptable salt, this based composition generally also include pharmaceutically acceptable carrier.This kind of load Body includes excipient well known in the art, such as diluent, adhesive, filler, disintegrant, lubricant, solvent, outstanding Floating agent, thickener, buffer, preservative.These compositions can be prepared according to method well known in the art.

The method that the present invention also provides the compound or its pharmaceutically acceptable salt that prepare formula (I).

Steroidal lactams (azepine-high steroidal) in the compound of formula (I) carries one on the ring of basic steroid backbone Or multiple amide functional groups.Known this kind of steroidal lactams can be closed by corresponding oximes and Beckmann rearrangement by ketone group steroidal Into (Koutsourea AI et al., Steroids, 2003,68 (7-8)：659-66；Mazur RH,J Org Chem,1963,28 (1)：248-250；Morzycki JW et al., Bioorg Med Chem, 1996,4 (8)：1209-15；Camoutsis C and Catsoulacos P,J Heterocycl Chem,1983,20(4)：1093-4；Huang Y et al., Molecules, 2013, 18(7)：7436-47).

The conventional method of Beckmann rearrangement

Oximes (1mmol) is dissolved in 17.5mL anhydrous twoAlkane.The mixture is cooled to 0 DEG C, thionyl chloride is added dropwise (1.9mL).The mixture is reached room temperature, stir 24 hours.Use NaHCO₃Stop reaction, with ethyl acetate (3x 20mL) Extract the mixture.Dry organic layer (Na₂SO₄), it is concentrated under reduced pressure, obtains crude product, passes through SiO₂This is further purified in chromatography Crude product.

Double (2- chloroethyls) amino-benzene oxygen propanoic derivatives of the compounds of this invention can be prepared as follows：

Substituted 3- (4- (double (2- chloroethyls) amino) phenoxy group) propionic acid has been synthesized since 4- nitrophenols.With not Same 3- chloropropionic acids are alkylated 4- nitrophenols, 3- (4-nitrophenoxy) propionic acid are obtained, by using H₂With Pd/C conducts Catalyst is further reduced into aminoderivative.Next, according to known methods, with oxirane in CH₃COOH、THF In make amino it is double-alkylation (Valu et al., J Med Chem, 1990,33 (11)：3014-19).Finally, by making in benzene Use POCl₃And heat and alcohol groups are changed into corresponding chloride, obtain corresponding 3- (4- (double (2- chloroethyls) amino) benzene Epoxide) propionic acid.In some cases, when amino or hydroxyl be present, the additional step for protection and deprotection reaction is must Need.For example, work as R₃It is NH₂Or during OH groups, respectively using Boc or Acetyl Protecting Groups (Valu KK et al., J Med Chem,1990,33(11):3014-9).By the same way, and since 2- nitrophenols or 3- nitrophenols, Ke Yihe Into the derivative of formula (I) compound of hydroxyl on the ortho position relative to amino or meta.

In order to produce steroidal lactams esters with alkylating agent, make the steroidal lactams comprising OH groups and DNA alkanisations Agent is reacted.For example, make steroidal lactams and 3- (4- (double (2- chloroethyls) amino) phenoxy group) propionic acid and DCC, DMAP or and 3- (4- (double (2- chloroethyls) amino) phenoxy group) propionyl chloride is mixed with 3- (4- (double (2- chloroethyls) amino) phenoxy group) propionic acid Anhydride reaction is closed, obtains corresponding esters.Any steroidal list lactams or double lactams can be derived using this method.

For being esterified the conventional method A of steroidal lactams

Alcohol (1mmol) is dissolved in 28mL anhydrous methylene chlorides.Then, sour (2mmol), DCC (2mmol) and catalytic amount are added DMAP (3mol%).After obtained solution is stirred 24 hours at room temperature, evaporation solvent is pure by Flash chromatography on Si-gel Change residue.

For being esterified the conventional method B of steroidal lactams

In round-bottomed flask, the 1mmol acid is diluted with 3.3mL anhydrous benzenes.Add 2,4,6- trichloro-benzoyl chlorides (1.2mmol) and triethylamine (2.4mmol), the mixture is flowed back 1 hour in an ar atmosphere.Described in being added into the mixture Solution of the steroidal alcohol 50mg (1mmol) in 3.3mL anhydrous benzenes, add the DMAP of catalytic amount.Persistently backflow 3 is small When.Benzene is removed completely by being evaporated in vacuo, and uses CH₂Cl₂Dilute remaining residue.Obtained with 5%HCl aqueous solution extractions mixed Compound, use 7%NaHCO₃The aqueous solution washs organic layer, is finally washed with water, uses Na₂SO₄Dry, removal of solvent under reduced pressure.Pass through silicon Glue flash chromatography carries out chromatography to residue.

For being esterified the conventional method C of steroidal lactams

By alcohol (1mmol), Et₃The DMAP of N (1.3mmol) and catalytic amount mixture is dissolved in CH₂Cl₂(5mL), Ran Houtian Add chlorobenzoyl chloride (0.12mL, 1.1mmol).Monitored and reacted by TLC, be stirred at room temperature 24 hours, be then dissolved in CH₂Cl₂, use Saturation NH₄The Cl aqueous solution is quenched.Organic layer is dried, passes through Flash chromatography on Si-gel purification of crude product.

The following examples are the illustrations of the present invention.

Embodiment 1

Scheme 1

1：By modifying (Camoutsis C and Catsoulacos P, J Heterocycl Chem, 1983,20 (4)： 1093-4) method, in 3 steps by testosterone 17- β-acetic acid Lipase absobed 3- azepines -17 beta-hydroxy-A- height -4_α- androstene -4- Ketone.Testosterone 17- β-acetic acid esters (914mg, 2.77mmol) is dissolved in 10ml anhydrous pyridines.Addition hydroxylamine hydrochloride (461mg, 6.64mmol), by the solution return stirring 6 hours.The solution is poured into water, the mixture (3x is extracted with ethyl acetate 30mL).Dry organic layer (Na₂SO₄), it is concentrated under reduced pressure, obtains crude product, passes through SiO₂The crude product is further purified in chromatography (eluant, eluent；Hexane-ethylacetate=4/1), obtain 675mg it is cis-and trans-oximes (74%), be white solid.

2：By it is cis-and trans-TESTOSTERONE ACETATE oximes (100mg, 0.145mmol) be dissolved in 3.5mL anhydrous two Alkane.The mixture is cooled to 0 DEG C, thionyl chloride (0.6mL) is added dropwise.The mixture is reached room temperature, stir 3 hours.With NaHCO₃Stop reaction, the mixture is extracted with ethyl acetate (3x 20mL).Dry organic layer (Na₂SO₄), it is concentrated under reduced pressure, Crude product is obtained, passes through SiO₂The crude product is further purified in chromatography (ethyl acetate), obtains -17 β of 63mg3- azepines-acetyl - 4 α of epoxide-A- height-androstene -4- ketone (63%), are white solid.

- 17 β of 3- azepines--4 α of acetoxyl group-A- height-androstene -4- ketone, be dissolved in 4.9mL MeOH by 1, be added dropwise LiOH (1N, 2mL).The mixture is stirred at room temperature 2 hours.Use NH₄Cl stops reaction, and it is mixed to extract this with dichloromethane (3x 10mL) Compound.Dry organic layer (Na₂SO₄), it is concentrated under reduced pressure, obtains-4 α of beta-hydroxy-A- height of 87mg 3- azepines-17-androstene-4- ketone 2, Yield is 74%.

3：- 4 α of beta-hydroxy-A- height of 3- azepines-17-androstene-4- ketone 2 is dissolved in 28mL anhydrous methylene chlorides.Then, add 3- (4- (double (2- chloroethyls) amino) phenoxy group) propionic acid (106mg, 0.573mmol), DCC (119mg, 0.574mmol) and urge The DMAP of change amount.After obtained solution is stirred 24 hours at room temperature, evaporation solvent, purified by Flash chromatography on Si-gel residual Excess (eluant, eluent；Hexane-ethyl acetate=1/2), obtain conjugate 3 (191mg, 99%) .3：Mp=53-56 DEG C；[α]D²³+ 10.5 (c=0.91CHCl₃)；¹H NMR(500MHz,cdcl₃) δ 6.92 (s, 1H), 6.83 (d, J=8.8Hz, 2H), 6.66 (d, J=8.6Hz, 2H), 5.72 (s, 1H), 4.66 (t, J=8.4Hz, 1H), 4.17 (t, J=6.0Hz, 2H), 3.63 (m, 4H), 3.59 (m, 4H), 3.32-3.04 (m, 2H), 2.75 (t, J=6.1Hz, 2H), 2.48 (m, 1H), 2.27 (m, 1H), 2.15 (m, 2H),1.50-1.98(m,10H),1.33(m,2H),1.14(s,3H),1.05(m,1H),0.80(s,3H)；¹³C NMR (126MHz,cdcl₃)δ171.0,170.4,161.3,151.3,140.8,118.8,116.3,114.4,82.7,64.4, 54.2,53.2,50.2,44.5,42.7,41.9,40.7,36.7,36.2,35.3,33.8,33.1,27.5,25.6,24.9, 23.4,21.3,12.1；FT-IR：3450,2925,1731,1651,1607,1512,1469,1353,1238,1181,1041, 869,813。

Embodiment 2

Scheme 2

4：According to previously described method synthesized female ketoxime (Ivanenko TI et al., Pharm Chem J, 1982,16 (10):751-6).Hydroxylamine hydrochloride is added into solution of the oestrone (100mg, 0.37mmol) in 2.2mL absolute ethyl alcohols (62mg, 0.88mmol) and pyridine (1.2mL).The mixture is flowed back 6 hours.Then water is added, with ethyl acetate (3x 10mL) extract the mixture.Dry organic layer (Na₂SO₄), it is concentrated under reduced pressure, obtains crude product, passes through SiO₂Chromatography is further Purify the crude product (eluant, eluent；Hexane:Ethyl acetate=3:1) the female ketoximes of 105mg (100%), are obtained, are white solid.

5：(Regan BM and Newton Hayes F, the J Am Chem of lactams 5 have been synthesized according to previously described method Soc,1956,78(3)：639-43).Female ketoxime (108mg, 0.376mmol) is dissolved in 6.3mL anhydrous twoAlkane.This is mixed Thing is cooled to 0 DEG C, and thionyl chloride (0.7mL) is added dropwise.The mixture is reached room temperature, stir 24 hours.Use NaHCO₃Make reaction Stop, the mixture is extracted with dichloromethane (3x 20mL).Dry organic layer (Na₂SO₄), it is concentrated under reduced pressure.Crude product is obtained, is led to Cross SiO₂Crude product (the eluant, eluent is further purified in chromatography；Hexane:Ethyl acetate=2:1) (the base of 42mg lactams 5, is obtained In the raw material of recovery, 56%), with the raw material [32mg raw materials (0.112mmol)] of recovery.

6：Lactams 5 is dissolved in 14mL dry DMFs.Then, 3- (4- (double (2- chloroethyls) amino) phenoxy group) third is added Sour (90mg, 0.293mmol), DCC (61mg, 0.293mmol) and catalytic amount DMAP.Obtained solution is stirred at room temperature After 24 hours, evaporation solvent, residue (eluant, eluent is purified by Flash chromatography on Si-gel；Dichloromethane/acetone=2/1), obtain To conjugate 6 (56mg, 68%) conjugates 6：[α]D²³+ 73.5 (c=0.90CHCl₃)；¹HNMR(500MHz,CDCl₃)δ7.25 (d, J=6.0Hz, 1H), 6.89 (d, J=8.8Hz, 2H), 6.82 (s, 1H), 6.68 (d, J=8.8Hz, 2H), 6.31 (s, 1H), 4.30 (t, J=6.1Hz, 2H), 3.62 (dt, J=29.2,6.6Hz, 8H), 2.97 (dd, J=15.1,9.0Hz, 2H), 2.88(m,2H),2.58-2.36(m,4H),2.23-2.00(m,2H),1.92-1.66(m,3H),1.60-1.29(m,4H), 1.19(s,3H)；¹³C NMR(126MHz,CDCl₃)δ171.7,169.8,151.3,148.5,141.0,137.8,137.2, 126.1,121.3,118.7,116.5,114.5,64.4,54.4,54.2,46.6,43.4,40.7,39.9,38.9,34.9, 30.5,29.5,26.5,25.9,22.1,19.8；FTIR：3329,2927,2850,1757,1626,1577,1512,1437, 1311,1244,1157,1088,1045,892。

Embodiment 3

Scheme 3

7：17- hydroxyl hero -4- alkene -3,11- diketone (484mg, 1.59mmol) is dissolved in 2.2mL acetic anhydrides.Then 4mg is added (0.037mmol) DMAP and 0.25mL anhydrous pyridines.The mixture is stirred at room temperature 24 hours.Stop reaction with water, use Ethyl acetate (3x 30mL) extracts the mixture.Dry organic layer (Na₂SO₄), it is concentrated under reduced pressure, obtains crude product, passes through SiO₂ Crude product (the eluant, eluent is further purified in chromatography；Hexane:Ethyl acetate=6:1) 472mg17- acetoxyl group heros -4-, is obtained Alkene -3,11- diketone, yield 86%.7：Mp=162-164 DEG C [α]_D ²³+148.0(c 1.68CHCl₃)；¹H NMR(500MHz, cdcl₃) δ 5.69 (s, 1H), 4.76 (t, J=8.6Hz, 1H), 2.83-2.69 (m, 1H), 2.54-2.20 (m, 6H), 2.01 (d, J=1.2Hz, 3H), 1.92 (m, 3H), 1.85-1.55 (m, 4H), 1.51-1.41 (m, 1H), 1.44-1.34 (m, 3H), 1.32- 1.10(m,2H),0.85-0.69(m,3H)；¹³C NMR(126MHz,cdcl₃)δ208.3,199.5,170.8,168.3, 124.6,80.2,62.6,54.8,49.4,46.2,38.2,37.0,34.7,33.7,32.1,31.7,27.6,22.9,20.9, 17.2,12.8；FT-IR：3443,2958,2935,2850,1732,1702,1677,1618,1426,1373,1360,1343, 1271,1238,1224,1045,1027

8：To solution of the 17- acetoxyl group hero -4- alkene -3,11- diketone (465mg, 1.35mmol) in 7mL absolute ethyl alcohols Middle addition hydroxylamine hydrochloride (100mg, 1.44mmol) and anhydrous pyridine (4.2mL).The mixture is stirred at room temperature 24 hours. Then, water is added, the mixture (3x 40mL) is extracted with ethyl acetate.Dry organic layer (Na₂SO₄), it is concentrated under reduced pressure, obtains thick Product, pass through SiO₂Crude product (the eluant, eluent is further purified in chromatography；Hexane:Ethyl acetate=20:1) 461mg oximes, are obtained Class 8 (95%).

9：Oxime 8 (264mg, 0.74mmol) is dissolved in 13mL anhydrous twoAlkane.The mixture is cooled to 0 DEG C, is added dropwise sub- Chlorosulfuric acid (1.4mL).The mixture is reached room temperature, stir 24 hours.Use NaHCO₃Stop reaction, with ethyl acetate (3x 20mL) extract the mixture.Dry organic layer (Na₂SO₄), it is concentrated under reduced pressure.Crude product is obtained, passes through SiO₂Chromatography is further Purify the crude product (eluant, eluent；Ethyl acetate:Methanol=1：0.03) 163mg lactams 9, yield 62%, are obtained.9：¹H NMR(500MHz,cdcl₃) δ 6.39 (s, 1H), 5.75 (s, 1H), 4.78 (t, J=8.6Hz, 1H), 3.35-3.18 (m, 1H), 3.09 (dt, J=14.7,7.2Hz, 1H), 2.67 (dd, J=14.9,8.2Hz, 1H), 2.48 (td, J=13.6,3.9Hz, 1H), 2.34-2.20 (m, 3H), 2.14 (dd, J=9.3,6.5Hz, 1H), 2.03 (s, 3H), 2.01-1.86 (m, 2H), 1.83- 1.53(m,5H),1.48-1.37(m,1H),1.38(s,3H),1.28-1.08(m,1H),0.76(s,3H)；¹³C NMR (126MHz,cdcl₃)δ209.0,170.8,169.5,158.4,120.1,80.1,62.4,55.1,49.9,46.7,43.6, 40.4,36.8,36.8,35.5,33.2,27.6,22.8,21.1,20.9,12.8；FT-IR：3428,2971,2920,2878, 2364,2341,1736,1701,1664,1639,1599,1444,1375,1339,1245,1127,1089,1046。

10：The 76mg of lactams 9 (0.21mmol) is dissolved in 3mL MeOH, LiOH (1N, 1.2mL) is added dropwise.By the mixture It is stirred at room temperature 1 hour.Use NH₄Cl stops reaction, and the mixture is extracted with dichloromethane (3x 5mL).Dry organic layer (Na₂SO₄), it is concentrated under reduced pressure, obtains 67mg lactams 10.10：¹H NMR(500MHz,dmso)δ7.72(s,1H),5.51(s, 1H), 4.66 (d, J=4.7Hz, 1H), 3.66 (dd, J=13.4,8.4Hz, 1H), 3.08-2.90 (m, 2H), 2.47-2.32 (m, 2H), 2.29 (d, J=11.5Hz, 1H), 2.21 (d, J=11.2Hz, 1H), 2.14-2.01 (m, 2H), 2.01-1.78 (m, 3H),1.74-1.50(m,3H),1.40(m,1H),1.28(s,3H),1.23(s,1H),1.15-1.02(m,1H),0.55(s, 3H)；¹³C NMR(126MHz,dmso)δ210.2,167.8,157.3,120.3,78.1,61.0,54.5,48.8,47.1, 43.1,40.4,36.8,35.5,34.9,33.1,29.9,22.3,20.9,11.8；FT-IR：3423,3262,2952,2923, 2853,1693,1647,1609,1458,1407,1375,1353,1261,1062。

11：Lactams 10 is dissolved in the anhydrous DCM of 8.2mL.Then, 3- (4- (double (2- chloroethyls) amino) phenoxy group) is added The DMAP of propionic acid (51mg, 0.17mmol), DCC (51mg, 0.25mmol) and catalytic amount.Obtained solution is stirred at room temperature After 24 hours, evaporation solvent, residue (eluant, eluent is purified by Flash chromatography on Si-gel；Ethyl acetate), obtain conjugate 11 (48.5mg, 96%).Conjugate 11：¹H NMR(500MHz,cdcl₃) δ 6.83 (d, J=9.0Hz, 2H), 6.67 (d, J= 9.0Hz, 2H), 6.11 (s, 1H), 5.76 (s, 1H), 4.86 (t, J=8.6Hz, 1H), 4.17 (t, J=6.2Hz, 2H), 3.61 (m,8H),3.25(m,1H),3.18-3.01(m,1H),2.84-2.59(m,2H),2.59-2.38(m,1H),2.37-2.25 (m,3H),2.16(m,1H),2.10-1.89(m,3H),1.87-1.55(m,4H),1.39(s,3H),1.26(m,2H),1.12 (m,1H),0.76(s,3H)；¹³C NMR(126MHz,cdcl₃)δ208.9,170.9,169.5,158.7,151.3,140.9, 119.9,116.2,114.5,80.4,64.2,62.4,55.1,54.3,49.9,46.8,43.6,40.7,36.8,35.5, 34.9,33.9,27.6,25.6,24.9,22.8,21.2,12.8；FT-IR：3432,3328,2927,2850,1733,1701, 1664,1626,1599,1513,1444,1389,1369,1310,1273,1243,1179,1087,1041,999。

Embodiment 4

Scheme 4

13：In sealing test tube azanol is being added into solution of 12 (100mg, the 0.28mmol) in 1.5mL absolute ethyl alcohols Hydrochloride (21mg, 0.31mmol) and anhydrous pyridine (0.9mL).The mixture is heated 7 days at 140 DEG C.Then, water is added, The mixture is extracted with ethyl acetate (3x 5mL).Dry organic layer (Na₂SO₄), it is concentrated under reduced pressure, obtains crude product, do not enter one The crude product is used for next step by step purifying.

14：Above-mentioned rough oximes 13 (0.28mmol) is dissolved in 4.9mL anhydrous twoAlkane.The mixture is cooled to 0 DEG C, thionyl chloride (0.54mL) is added dropwise.The mixture is reached room temperature, stir 24 hours.Use NaHCO₃Stop reaction, use second Acetoacetic ester (3x 20mL) extracts the mixture.Dry organic layer (Na₂SO₄), it is concentrated under reduced pressure.Crude product is obtained, passes through SiO₂Color Crude product (the eluant, eluent is further purified in spectrometry；Ethyl acetate:Methanol=1：0.1) 52mg lactams, is obtained, yield is 50%.14：¹H NMR(500MHz,cdcl₃) δ 7.00 (s, 1H), 5.77 (s, 1H), 5.59 (s, 1H), 4.61 (t, J=8.3Hz, 1H), 3.20 (m, 2H), 3.02 (dd, J=9.6,5.0Hz, 1H), 2.48-2.40 (m, 2H), 2.31 (d, J=13.7Hz, 1H), 2.16(m,2H),2.09-2.0-1.97(m,5H),1.74-1.84(m,2H),1.51-1.40(m,2H),1.35-1.30(m, 1H),1.24(s,3H),1.23(m,1H),1.08(m,1H),0.95(s,3H)；¹³C NMR(126MHz,cdcl₃)δ175.1, 170.9,169.4,156.3,120.4,80.1,64.2,55.5,45.2,44.6,41.0,40.8,38.0,36.3,34.4, 31.0,25.4,25.2,21.9,21.0,11.7。

15：The 28mg of lactams 14 (0.084mmol) is dissolved in 1.2mL MeOH, LiOH (1N, 0.5mL) is added dropwise.This is mixed Compound is stirred at room temperature 1 hour.Use NH₄Cl stops reaction, and the mixture is extracted with ethyl acetate (3x 5mL).Drying is organic Layer (Na₂SO₄), it is concentrated under reduced pressure.Pass through SiO₂Crude product (eluant, eluent is further purified in chromatography；Ethyl acetate:Methanol=1： 0.1) 28mg lactams 15, yield 100%, are obtained.15：¹H NMR(500MHz,dmso)δ7.75(s,1H),6.13(d,J =3.9Hz, 1H), 5.53 (s, 1H), 4.66 (d, J=5.3Hz, 1H), 3.40 (m, 2H), 3.11-2.93 (m, 2H), 2.44- 2.34(m,1H),2.29(s,2H),2.04-1.72(m,5H),1.65(m,2H),1.24(m,3H),1.20(s,3H),0.89 (m,1H),0.66(s,3H)；¹³C NMR(126MHz,dmso)δ174.6,167.7,156.1,120.1,78.1,69.8,63.4, 44.9,44.2,41.2,40.4,37.7,35.3,33.7,31.3,27.7,24.4,20.9,10.7。

16：Lactams 15 (30mg, 0.09mmol) is dissolved in the anhydrous DCM of 9mL.Then, 3- (4- (double (2- chloroethenes are added Base) amino) phenoxy group) and propionic acid (67mg, 0.22mmol), DCC (60mg, 0.29mmol) and catalytic amount DMAP.At room temperature After obtained solution is stirred 24 hours, evaporation solvent, residue (eluant, eluent is purified by Flash chromatography on Si-gel；Acetic acid second Ester/MeOH=10/1), obtain conjugate 16 (39mg, 70%).¹H NMR(500MHz,cdcl₃) δ 6.85 (d, J=9.0Hz, 2H), 6.76 (s, 1H), 6.65 (d, J=9.0Hz, 2H), 5.79 (s, 1H), 5.46 (s, 1H), 4.68 (t, J=8.3Hz, 1H), 4.17 (t, J=6.2Hz, 2H), 3.60 (m, 4H), 3.51-3.42 (m, 1H), 3.18 (m, 2H), 3.02 (dd, J=9.5, 5.0Hz 1H), 2.85-2.67 (m, 2H), 2.51 (d, J=13.8Hz, 1H), 2.44 (dd, J=13.6,10.1Hz, 1H), 2.33 (d, J=13.8Hz, 1H), 2.25-2.06 (m, 2H), 2.07-1.73 (m, 5H), 1.73-1.28 (m, 5H), 1.25 (s, 3H),1.17-1.03(m,2H),0.96(s,3H)；¹³C NMR(126MHz,cdcl₃)δ174.7,170.9,169.1,155.6, 151.3,140.9,120.7,116.3,114.4,80.5,64.2,64.1,55.5,54.2,45.3,44.5,41.1,40.7, 38.1,36.3,34.8,34.4,33.9,31.0,25.6,25.5,25.2,24.9,21.9,11.8.FT-IR：3410,3330, 2926,2850,1734,1654,1627,1577,1513,1445,1349,1273,1243,1180,1133,1110,1087, 1044,890。

Embodiment 5

Scheme 5

According to Koutsourea et al. (Steroids, 2003,68 (7-8)：Lactams 17 659-66) is synthesized.

18：In round-bottomed flask, 48mg (0.157mmol) described acid is diluted with 0.5ml anhydrous benzenes.Add 2,4,6- trichlorines Chlorobenzoyl chloride (30 μ l, 0.189mmol) and triethylamine (53 μ l, 0.378mmol), the mixture is flowed back in an ar atmosphere 1 small When.Solution of the steroidal alcohol 50mg (0.157mmol) in 0.5ml anhydrous benzenes, and the 4- of catalytic amount are added into the mixture Dimethylamino naphthyridine.Persistently flow back 3 hours.Benzene is removed completely by being evaporated in vacuo, and uses CH₂Cl₂Dilute remaining residue.With The mixture that 5%HCl aqueous solution extractions obtain, uses 7%NaHCO₃The aqueous solution washs organic layer, is finally washed with water, uses Na₂SO₄ Dry, removal of solvent under reduced pressure.Chromatography (eluant, eluent is carried out to residue on a silica gel column；Ethyl acetate/MeOH=100/1), Obtain 46mg conjugates 18, yield 48%.Conjugate 18：¹H NMR(500MHz,cdcl₃) δ 6.84 (d, J=8.5Hz, 2H), 6.67 (d, J=8.5Hz, 2H), 5.90 (s, 1H), 5.86 (s, 1H), 4.78 (m, 1H), 4.19 (m, 2H), 3.58-3.59 (m, 8H),3.48(m,1H),

2.61-1.25(18H),1.29(s,3H),0.88(s,3H)；[M+H]⁺=605.

Embodiment 6

Scheme 6

According to Koutsourea et al. (Steroids, 2003,68 (7-8):Lactams 19 659-66) is synthesized.

20：In round-bottomed flask, 46mg (0.15mmol) described acid is diluted with 0.5ml anhydrous benzenes.Add 2,4,6- trichlorines Chlorobenzoyl chloride (28 μ l, 0.18mmol) and triethylamine (50 μ l, 0.36mmol), the mixture is flowed back 1 hour in an ar atmosphere. Solution of the steroidal alcohol 50mg (0.150mmol) in 0.5ml anhydrous benzenes, and the 4- diformazans of catalytic amount are added into the mixture Aminopyridine.Persistently flow back 3 hours.Benzene is removed completely by being evaporated in vacuo, and uses CH₂Cl₂Dilute remaining residue.With 5% The mixture that HCl/water solution is obtained by extraction, uses 7%NaHCO₃The aqueous solution washs organic layer, is finally washed with water, uses Na₂SO₄It is dry It is dry, removal of solvent under reduced pressure.Chromatography (eluant, eluent is carried out to residue on a silica gel column；Ethyl acetate/MeOH=100/2), obtain To 19mg conjugates 20, yield 20%.20：¹H NMR(500MHz,cdcl₃) δ 7.18 (s, 1H), 6.84 (d, J=8.5Hz, 2H), 6.65 (d, J=8.5Hz, 2H), 6.60 (s, 1H), 5.82 (s, 1H), 4.80 (1H, m), 4.21 (2H, m), 3.50 (m, 8H),3.20(1H,m),2.80-1.30(19H),1.20(s,3H),0.9(s,3H)；[M+H]⁺=621.

Embodiment 7

Scheme 7

According to Koutsourea et al. (Steroids, 2003,68 (7-8):Lactams 21 659-66) is synthesized.

22：In round-bottomed flask, 37mg (0.12mmol) acid is diluted with 0.4ml anhydrous benzenes.Add 2,4,6- trichloro-benzenes first Acyl chlorides (22 μ l, 0.144mmol) and triethylamine (40 μ l, 0.288mmol), the mixture is flowed back 1 hour in an ar atmosphere.To Solution of the steroidal alcohol 50mg (0.120mmol) in 0.4ml anhydrous benzenes, and the 4- diformazan ammonia of catalytic amount are added in the mixture Yl pyridines.Persistently flow back 3 hours.Benzene is removed completely by being evaporated in vacuo, and uses CH₂Cl₂Dilute remaining residue.Use 5%HCl The mixture that aqueous solution extraction obtains, uses 7%NaHCO₃The aqueous solution washs organic layer, is finally washed with water, uses Na₂SO₄Dry, Removal of solvent under reduced pressure.Chromatography (eluant, eluent is carried out to residue on a silica gel column；Ethyl acetate), 34mg conjugates 22 are obtained, Yield is 40%.22：¹H NMR(500MHz,cdcl₃) δ 6.84 (d, J=8.5Hz, 2H), 6.60 (d, J=8.5Hz, 2H), 5.90(s,1H),5.79(s,1H),4.80(m 1H),4.15(m,2H),3.5(m,8H),3.25(m,1H),2.8-0.8 (22H)；[M+H]⁺=605.

Embodiment 8

The in vitro and in vivo biology test of active anticancer

A)Anticancer Activity in vitro

In order to test cell inhibiting caused by the compound newly synthesized and cytotoxic activity, 9 are handled and have fully built Vertical human carcinoma cell line's (table 1).The cell line derives from American type culture collection (ATCC), and according to operation instruction It is set to be grown in different culture media.MTT (survey by (3- (4,5- dimethylthiazole -2- bases) -2,5- diphenyltetrazolium bromides) The method of determining is the method for the abundant foundation and standard of the cell inhibitory activity and cytotoxic activity for evaluating medicine and chemicals (Trafalis DT et al., J BUON, 2003,8:333-9；Trafalis DT et al., J BUON, 2004,9 (3):275-82； Trafalis DT et al., J BUON, 2005；10:227-34；TrafalisDT et al., Breast Cancer Res Treat, 2006,97：17-31).In short, by cell with every hole -3 × 10⁴The density of individual cells/ml on 96 orifice plates bed board and In 5%CO at 37 DEG C₂Maintained 72 hours in incubator, and it is grown with individual layer or suspension formation.After 24 hours, 0.1- is used 100 μm of ol/l compound processing cell 48 hours, as described above with MTT (Sigma, St Louis, Missouri, USA) generations Thank to the viability of determination method estimation culture cell.On ELISA readers (Versamax, Orleans, USA), in 540nm ripples The absorbance of the dyestuff of strong point measurement conversion.Produce the every of 50% or complete (100%) growth inhibition (being respectively GI50 and TGI) The mean concentration of kind of medicine and [(half-maximal cell poison is dense to 50% drug concentration of culture cell generation cytotoxicity Degree (IC50)] calculated using linear regression method.Use 7 absorbance measurements [24 hours time (Ct24), control growths Test vector generation for testing IC (Tt72x) in the presence of the medicine of 72 hours (Ct72) and 5 kinds of concentration levels], calculate every kind of drug concentration level Under growth percentage.According to National Cancer Institute (National Cancer Institute) (NCI) by growth inhibition hundred Ratio is divided to be calculated as：[(Tt72x)-(Ct24)/(Ct72)-(Ct24)] x 100, for Tt72x>Ct24 concentration, and [(Tt72x)-(Ct24)/Ct24] x 100, for Tt72x<Ct24 concentration；By [(Tt72x)-(Ct24)/(Ct72)- (Ct24)] x 100=50 calculate GI50；TGI is calculated by [(Tt72x)-(Ct24)/(Ct72)-(Ct24)] x 100=0；And by [(Tt72x)-(Ct24)/Ct24] x 100=50 calculate IC50.All experiments are triplicate to be carried out.

Table 1

The in-vitro cell growth that test compound is induced human carcinoma cell line suppresses (GI50, TGI) and cytotoxicity (IC50) result of effect is as shown in table 2,3,4.

Table 2

Table 3

Table 4

B)Internal acute toxicity

For intraperitoneal (i.p.) processing, the preceding stock solution for preparing test compound at once is being used.Initially it is being dissolved in After 10% dimethyl sulfoxide (DMSO), they are suspended in corn oil with desired concentration.This concentration will not produce in itself can It was observed that toxic action.

C57BI/6 female mices are used for toxicity research.Mouse derives from Hellenic Pasteur Institute experiment Department.

In short, such as (Catsoulacos P et al., the Cancer Chemother fully described before this Pharmacol,1979,3(1):67-70；Catsoulacos P et al., J Pharm Sci, 1978,67 (9):1342-3； Catsoulacos P et al., Anticancer Res, 1995；15:827-30), with 4 various dose single intraperitoneals (i.p.) after injecting the group of ten (10) C57BI/6 mouse, the acute toxicity of test compound induction is determined；Observe mouse 30 My god, and estimate that (30- days curves) determines the therapeutic dose of compound afterwards in diagram, it is generally defined as LD10 (lethal dose of 10% animal) and LD50 (lethal dose of 50% animal).According to lethal in C57BI/6 mouse Rate have rated the toxicity of test compound.LD50 and LD10 values are estimated with diagram method, wherein because the toxicity of each dosage causes Percent mortality be shown in ordinate, and institute's applied dose is shown (table 5) on the horizontal scale.

C) internal antitumor activity

According to National Cancer Institute (NCI), USA scheme, 10 were implanted into by intraperitoneal (i.p.) at the 0th day⁶It is individual The ascites cells of P388 lymphocytic leukemias start to test.For i.p. processing, it is prepared for testing chemical combination at once using preceding The stock solution of thing.Antitumor activity have rated according to knurl parameter T/C% processed, the knurl parameter T/C% processed means drug therapy Mean survival time (MST) of the animal (T) compared with the control (C) of saline treatment.According to NCI (USA), active lowest bid Standard is that T/C is higher than 125%.In addition, antitumor activity (is cured according to the quantity survey of long-term survivors：It is defined as in tumour The mouse to be survived 90 days after inoculation) (Golidim A et al., Nat Cancer Inst Monogr, 1980,55：25-26；NCI Monograph, NIH publication 1986,55:80-193).

BALB/c severe combined immunodeficients female mice is used for antitumor evaluation.These animals are taken in BALB/c backgrounds Band severe combined immunodeficient mutation (scid), and from NCSR " Demokritos ", biological study is obtained.Mouse is kept Under conditions of constant temperature and humidity, in having the sterile cage of water and food.Each treatment group includes 6 mouse, and control group includes 8 Mouse.

The therapeutic dose of tested compound is defined with corresponding LD10 (mg/kgr).

Acute toxicity of the compound of table 5. in C57BI/6.

50% and 10% lethal dose of mouse population treated LD50 and LD10=.

Compound	LD50(mg/kg)	LD10(mg/kg)
			pBCEAPOPA	20	15
ASE	50	30
			3	150	130
6	100	80
			18	110	85
20	135	110
			22	-	>300
11	130	100
			16	165	140

The compound that table 6. is tested is to anti-leukocythemia liveness inside mouse P388 lymphocytic leukemias.

*p<0,001

BALB/c severe combined immunodeficients female mice is used for body of the tested compound to human ovarian cancer SCOV-3 Interior antitumor evaluation.Right veutro or left veutro subcutaneous vaccination 3 × 10 in every animal⁶The suspension of individual SCOV-3 cancer cells/ 0.2 milliliter/mouse.Mouse is maintained under conditions of constant temperature and humidity in having the sterile cage of water and food.Each treatment group and Control group includes 10 mouse.Lab scenario according to fully having established is tested.According to tumour cell dynamics And biological characteristics, by treated animal (T) relative to the gross tumor volume of control (C) mean change (T/C%=TI, Tumor suppression) and the middle position time-to-live increase, it is determined that the effect of medicine.Gross tumor volume or weight are calculated as 0.52x a²X b, wherein a and b are secondary and main tumour axles, and with the standard error of mean tumour volume ± average value after treatment Poor (± SEM) maps on semilog schematic diagram data to the time.When tumour reaches 0.085-0.1mm³Volume when, will be small Mouse is divided into control group and medication therapy groups (10 mouse/groups), has similar mean tumour volume in every group.The 1st day, Tested compound is applied with LD10/4 dosage i.p. respectively within 5 days and the 9th day.

In order to evaluate antitumous effect, (a) determines average tumor weight weekly or mean tumour volume change, and leads to Cross below equation and calculate tumor suppression (TI)：TI (%)=and [1) (TWT) TWZ)/(TWC) TWZ)] × 100, wherein TWT is true Tumor weight (mg) or the gross tumor volume (mm being set in the animal treated when evaluation³), TWZ is determined to be in treatment and opened The tumor weight (mg) or gross tumor volume (mm of (when zero or the 1st day) when beginning³), TWC be determined to be in evaluation when without controlling Tumor weight (mg) or gross tumor volume (mm in the animal (control) for the treatment of³), the percentage of (b) in the 70th day survivor (OS%), the percentage (PFS%) of (c) in the 70th day survivor without tumour progression.As a result as shown on table 7.

The compound that table 7. is tested is to antitumor activity inside SCOV-3 human ovarian cancers

All differences on TI%, OS%, PFS% are in p<It is significant in 0.05-0,001 level

D) pharmacotoxicological effect

Described new lactams steroidal alkylating agent is induction of to the notable of poly- (ADP- ribose) polymerase (PARP1/2) activity Inhibitory action, the half maximum suppression concentration (IC50) less than 1.7 μ Μ is shown, better than well-known PARP1/2 inhibitor 3-AB (3-AB).In addition, described new lactams steroidal alkylating agent in vitro and in vivo with dosage and time according to Transcription and mRNA expression of the property mode to PARP1 and PARP2 is relied to generate significant change.Originally or with relatively low dosage, it PARP1 and PARP2mRNA can be induced to express increase, it reaches higher than 5-400 times of control value so that intracellular NAD+ concentration with Cell ATP, which is exhausted, produces change, and later or under higher dosage, they induce PARP1 and PARP2mRNA expression to reduce, its It can reach close to 100%.By Sister chromatid exohange (SCE) determination method and in vivo by being produced in serum or urine 8- hydroxyl -2'- deoxyguanosines (8-OHdG) adduct is evaluated, and described new lactams steroidal alkylating agent generates significant DNA Damage, the DNA damage is suitable with the DNA damage that their alkanisation composition individually induces, while they generate it is significantly higher Antitumor activity.In addition, described new lactams steroidal alkylating agent significantly suppress (>60%) ERK1/2 and AKT1/2 phosphorus Acidifying, and therefore significantly suppress the activation of PI3K and MAPK molecular signal pathways.It has extensively studied first described interior The molecular pharmacology effect of acid amides steroidal alkylating agent.

Claims

1. the compound or its pharmaceutically acceptable salt of formula (I)

Wherein

R₁Selected from the group being made up of following group

R₃Selected from by H ,-OH ,-NH₂The group of composition.

2. compound according to claim 1 or its pharmaceutically acceptable salt, wherein R₁Selected from the group being made up of following group

3. compound according to claim 2 or its pharmaceutically acceptable salt, wherein R₁Selected from the group being made up of following group

4. compound according to claim 2 or its pharmaceutically acceptable salt, wherein R₁It is selected from

5. according to any one of claim 1-4 compound or its pharmaceutically acceptable salt, wherein R₂Selected from by H ,-CH₃、-CH =CH₂、-CH₂-CH₃、-CH₂CH₂CH₃The group of composition.

6. compound according to claim 5 or its pharmaceutically acceptable salt, wherein R₂It is H.

7. according to any one of claim 1-6 compound or its pharmaceutically acceptable salt, wherein R₃It is H.

8. according to any one of claim 1-6 compound or its pharmaceutically acceptable salt, wherein R₃It is-NH₂。

9. according to any one of claim 1-8 compound or its pharmaceutically acceptable salt, it is used in medicine.

10. according to any one of claim 1-8 compound or its pharmaceutically acceptable salt, it is used in the treatment of cancer.

11. according to any one of claim 1-8 compound or its pharmaceutically acceptable salt, it be used for oophoroma, breast cancer, In the treatment of prostate cancer or leukaemia.

12. pharmaceutical composition, it includes the compound or its pharmaceutically acceptable salt and medicine according to any one of claim 1-8 Acceptable carrier on.